three techniques, one system: how to effectively characterize complete muscle function
TRANSCRIPT
Three Techniques, One System: How to Effectively Characterize Complete Muscle Function
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Three Techniques, One System: How to Effectively Characterize Complete Muscle Function
1. A Brief History Of Aurora Scientific And The 3-in-1 (M. Borkowski)
2. Muscle Function Assays: In-vivo, In-situ, In-vitro (R. Khairallah)
3. Understanding Functional Assessments (C. Ward)
4. How To Choose The Correct Assays Based On Your Objectives (C. Ward)
5. Q&A Session (Group)
Sponsored by:
InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in
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Three Techniques, One System: How to Effectively Characterize Complete Muscle Function
Matt Borkowski
Sales & Support Manager
Aurora Scientific
About Aurora Scientific
• Aurora has served the muscle community for nearly 20 years.
• Test systems and solutions ranging from single cells up to the whole animal.
Cell
Whole Animal
Fiber
Whole Muscle
Ramzi Khairallah Co-Founder, President • Founded in 2014
• Contract Research Organization providing:
Neuromuscular and muscle phenotyping in pre-clinical models
Assay development, study design
Training
Myologica can validate your disease model, screen your compounds, or provide insight into the mechanisms at work in your system.
In partnership with:
Christopher Ward Co-Founder, Scientific Director
Asst. Prof. University of Maryland
Introduction: Neuromuscular Phenotyping
• In the rodent, measures of physical performance are of growing interest.
• Many laboratories have adopted assays that provide gross assessments of functional performance.
• While these are informative screening assays, the functional phenotypes are not specific to neuromuscular function.
Voluntary Running Wheel
Forced Treadmill Exercise Capacity
Grip-strength
Determinants of Neuromuscular Performance
Muscle Fibers
Assays with Behavioral Component
• Treadmill
• Running Wheel
• Grip Strength
Aurora 3 in 1
Aurora Single Cell Setup
In situ In vitro In vivo
The 3-in-1 System
• System is based around our Dual Mode Lever
• Single instrument for isolated muscle or whole animal
– Lever arm for ex-vivo & in-situ
– Footplate for in-vivo.
• Goes light years beyond isometric
– Power, Force-Velocity, Work Loops and many other protocols trivial to perform.
Experimental Apparatus
• Temperature controlled apparatus for various animal models.
• Fine positioning of transducer relative to animal or muscle.
• Customized clamps and accessories designed to make switching easy.
Experimental apparatus for rat. In-vivo
measurement configuration shown.
System Control & Software
• All experiments performed with our customized acquisition software (Dynamic Muscle Control).
• Stimulation, stretches, slacks and muscle tension all fully controlled through software.
• Library of standard protocols allows for infinite customization.
• Straightforward to use. Once muscle/animal attached, load protocol and press start.
System Control & Software
Data Analysis Data Analysis
• Software suite for performing visualization and analysis
• Automatic High Throughput data analysis module: Automated analysis of fatigue, force-frequency, force-velocity
• Available programming service for customer specific features and free updates released regularly
Who is this system useful for?
Muscle Physiologists
Exercise Scientists
Metabolic & Cardiovascular Scientists
Bioengineers & Biologists
Geneticists
Neuroscientists
Pharmacologists & Biochemists
Anyone studying muscle mechanics
Three Techniques, One System: How to Effectively Characterize Complete Muscle Function
Ramzi Khairallah
Co-Founder, President Myologica
The 3-in-1 System
In situ In vitro In vivo
In vivo – Ankle Torsion
Photo courtesy of Files Lab; Wake Forest University
Mouse Dorsiflexor Torque Assay
Ankle flexors (dorsiflexion) • Tibialis anterior
• Extensor digitorum longus (EDL)
Ankle extensors (plantarflexion) • Gastrocmenius
• Soleus
Note: Dr. Richard Lovering (Univ. Maryland School of Medicine) has developed a method to evaluate the quadriceps muscle (i.e. knee extension) with a custom mechanical mounted to the torque sensor.
In vivo : Without surgical isolation of the muscle or alteration of the neurovascular supply. The mouse or rat is deeply anesthetized
The hind limb is mechanically stabilized
The foot is secured in a foot-plate mounted to the torque sensor.
Percutaneous or subcutaneous stimulation of the motor nerve elicits muscle contraction.
Torque about the joint is measured
In vivo – Ankle Torsion
Torque is often normalized to adjust for muscle mass/animal size • Animal weight
• Muscle mass
• Estimates of muscle cross-sectional area
• Direct measures of muscle cross-sectional area
o MRI, CT scan
o Histology
Foot Plate (torque arm)
Force causing torque
τ = F x L where τ is the torque F is the force L is the length of the lever arm
In vivo - Ankle Torsion
Strengths Most high-throughput assay
Assay is across the joint which is the most
physiologically relevant
No surgical isolation of the muscle or alteration of the neurovascular supply.
Repeated measures (days, weeks) is possible.
Challenges Determination of “muscle specific”
contribution to function may be challenging
Dependent on intact neuromuscular junction
Placement of electrodes requires practice to achieve consistency and can be technically challenging
In vivo - Ankle Torsion
In vitro In vivo
The 3-in-1 System
In situ
Photo courtesy of Granzier Lab; University of Arizona
Mouse TA muscle under fatigue test
Ankle Dorsiflexors • Tibialis anterior
• Extensor digitorum longus (EDL)
Ankle Plantarflexors • Gastrocnemius
• Soleus
Trapezius, diaphragm, etc…
As this an invasive technique, it is best suited for terminal experiments.
In situ – Intact Muscle
Mouse TA muscle
• The mouse or rat is deeply anesthetized.
• The hind limb is mechanically stabilized and the skin retracted to expose the muscle group.
• The muscle of choice is partially dissected, the distal tendon is severed, and tied to the force transducer with a silk suture.
• Percutaneous or subcutaneous
stimulation of the motor nerve - or – muscle can be used to elicit muscle contraction.
In situ – Intact Muscle
Again, force is often normalized to adjust for muscle mass or cross-sectional area.
• Muscle mass
• Estimates of muscle CSA
• Direct measures of fiber CSA – Histology
In situ - Intact Muscle
Additional assays are possible
Motor Unit Number Estimation (MUNE)
Motor Unit: One neuron and all the muscle fibers it innervates • Sever the nerve, use suction
electrode to stimulate nerve evoked muscle contractions.
• With small steps in voltage, assay quantal increases in force production.
• Use predictive modeling to determine motor unit number.
In situ - Intact Muscle
Strengths While surgically invasive, the
neurovascular supply is still preserved.
Surgical isolation allows direct measure of a specific muscle’s contribution
Determination of “muscle specific” contribution to function is possible by direct muscle stimulation.
Challenges Invasive
Technically more difficult
Surgical preparation makes
temporal repeated measures challenging
In situ - Intact Muscle
The 3-in-1 System
In situ In vivo In vitro
• Muscle surgically excised from the animal
• “Classical” isolated muscle experiment
• Horizontal muscle chamber
• Tension & length recorded and controlled by one instrument
• Field stimulation of the muscle - or - nerve stimulation with suction electrode
Courtesy of Wilkinson Lab; SJSU
In Vitro
Courtesy of Wilkinson Lab; SJSU
EDL Muscle with nerve attached
• Muscle surgically excised from the animal
• “Classical” isolated muscle experiment
• Horizontal muscle chamber
• Tension & length recorded and controlled by one instrument
• Field stimulation of the muscle - or - nerve stimulation with suction electrode
In Vitro
Any small muscle whose metabolic needs can be met through diffusion.
Extensor digitorum longus (EDL)
Soleus
Lumbricalis
Diaphragm (hemi-diaphragm)
Heart:
– Papillary
– Trabeculae
Photo courtesy of Wilkinson Lab; SJSU
In Vitro
Strengths: Surgical excision allows direct measure
of muscle contractility independent of neuronal integrity.
Dissection of the muscle with a portion of the motor nerve allows ex vivo nerve-muscle function to be assessed.
Chemical/pharmacological manipulation possible
Challenges: Surgical excision negates blood flow
support.
Technically challenging
Prep viability is limited by oxygen/metabolic substrate diffusion to inner muscle fibers and and CO2 diffusion out from inner fibers.
Preparation makes temporal repeated measures impossible
In Vitro
Three Techniques, One System: How to Effectively Characterize Complete Muscle Function
Christopher Ward
Co-Founder, Scientific Officer Myologica
Asst. Professor
University of Maryland
Basic Assumptions
Optimal muscle length (i.e., resting length: L0) for each preparation.
Supra-maximal stimulation to ensure recruitment of all muscle fibers with single action potential (i.e., twitch) stimulation.
Note: Both L0 and stimulation intensity are determined for each preparation.
• Contractile Function
o Isometric Contractions
o Concentric Contractions
• Fatigue
• Injury Susceptibility
o Eccentric
o Isometric
Functional assays enabled by the 3-in-1 system
Functional assays enabled by 1300A Functional assays enabled by the 3-in-1 system
• Contractile Function
o Isometric Contractions
o Concentric Contractions
• Fatigue
• Injury Susceptibility
o Eccentric
o Isometric
Variety of variables can be examined to gain insight into muscle function
• Peak torque/force
• Kinetics
+df/dt
–df/dt
Time to max
Time to min
The most common functional assessment is isometric contraction – force produced with no change in muscle length.
Force vs. Stimulation Frequency
• Trains of action potentials at increasing frequency until there is no further force increase.
• The peak force responses for each stimulation frequency is determined.
Functional Assessments: Isometric Contraction
Force vs. Stimulation Frequency Relationship for EDL in vitro
0.2 0.4 0.6 0
5
10
15
20
25
30
35
Iso
met
ric
Ten
sio
n (
g)
Time (sec)
250 msec.Train
Pratt et. al., J Physiol, Volume 591, Issue 2 2012; Lovering Lab. UMB
Nerve Stimulation
• The femoral nerve is stimulated every second for 2 mins
• (330-ms tetanic)
Muscle Stimulation
• The electrodes are placed directly over the muscle
• A direct muscle tetanic contraction is superimposed every 15 s
Fatigue in situ
Functional Assessments: Isometric Contraction
Force v. Frequency
Cohen et. al., J Physiol, 2015; Wagner Lab. JHU
Functional Assessments: Isometric Contraction
Force v. Frequency
+dF/dT
-dF/dT
Contractile Kinetics
Functional Assessments: Isometric Contraction
Cohen et. al., J Physiol, 2015; Wagner Lab. JHU
Force v. Frequency
*
Functional Assessments: Isometric Contraction
Contractile Kinetics
+dF/dT
-dF/dT
Cohen et. al., J Physiol, 2015; Wagner Lab. JHU
Muscles of locomotion transmit force across a joint resulting in a change in muscle length and articulation of the joint.
Concentric contraction – Muscles actively shortening
Eccentric Contraction – Muscles actively lengthening
Popular type of contraction to study
• Much of a muscle's normal activity occurs while it is actively lengthening.
• Muscle injury and soreness are selectively associated with eccentric contraction
Functional Assessments
in vivo Eccentric Contractions - Susceptibility to Injury
mdx WT
0.1
N/C
M
100 msec
STIM
STRETCH
1st
20th
Khairallah et al. Science Sig. 2012
in vivo Eccentric Contractions - Susceptibility to Injury
Khairallah et al. Science Sig. 2012
1 sec
STIM
0.1
N/c
m
in vivo Eccentric Contractions - Susceptibility to Injury
Khairallah et al. Science Sig. 2012
How to choose the best assay(s)?
• The 1300A ‘3-in-1’ System provides the scientist with many options for the functional assessment of neuromuscular phenotype
• While it is tempting run all the assays possible, this is rarely feasible, time efficient, or cost-effective
What is the phenotyping goal?
Neuro Muscular
In vivo • Moderate throughput • Assay is across the joint which is the most
physiologically relevant • Repeated measures (days, weeks) is possible
In situ • Direct measure of a specific muscle
• Motor nerve or muscle stimulation
In vitro • An ex vivo prep allowing direct measure of muscle
contractility independent of neuronal integrity.
1300A ‘3-in-1’ System
Thank You!
For additional information on the Aurora 3-in-1 system, muscle measurement assays, and other products related to the study of muscle function please visit:
http://www.AuroraScientific.com/
InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information regarding innovative technologies, protocols, research
tools and laboratory services.